One of the primary goals of this laboratory is to improve the treatment of breast cancer in order to prevent the disease from recurring and develop strategies to overcome resistance to radiation (and chemotherapy). Our studies have demonstrated that 1,25-dihydroxy vitamin D3/1,25-D3 (the active fonn of vitamin D) or its analogs confer enhanced sensitivity to irradiation (and chemotherapy with Adriamycin) My research is focused on substantiating the role of 1,25-D3 and the vitamin D3 analog EB1089(EB) in radiosensitizing breast cancer cells and elucidating the autophagic signaling cascades involved in radiosensitization. The first specific aim is to substantiate that autophagy is the mode of radiation sensitization in breast tumor cells treated with EB or 1,25-D3. Pharmacological (3-methyl adenine, Bafilomycin, and Chloroquinone) and genetic approaches (shRNA against Beclin-1, ATG5 and ATG7) will be used to inhibit autophagy. Autophagy will be monitored via well-established methods including acridine orange staining, western blotting for LC3 cleavage, and electron microscopy. We expect that these studies may also show that suppression of proliferative recovery in irradiated cells by EB or 1,25-D3 will be reversed if autophagy is inhibited. The second specific aim is to determine if EB and 1,25 D3 can sensitize radioresistant cells, MCF-7 Her-2/neu and SKBR3 cells (that overexpress Her-2/neu) will be pretreated with 1,25-D3 or EB and cell viability will be analyzed by trypan blue exclusion and clonogenic survival assays. Cell death will be monitored by the TUNEL assay for apoptosis, acridine orange/LC3 cleavage for autophagy and micronuclei formation via DAPI staining for mitotic catastrophe. We expect that EB and 1,25- D3 will be shown to overcome resistance to radiation that occurs through overexpression of Her-2/neu. The third specific aim is to determine the involvement/modulation of the ER stress and mTOR pathways in radiosensitization of breast cancer cells. We propose to measure the impact of EB /1,25 D3 and radiation on the levels of crucial mediators involved in the ER stress (elF2a, JNK and XBP-1, PERK, IRE-1) and mTOR (4EBP-1 & S6k1) pathways in MCF-7, MCF-7 Her-2/neu and SKBR3 cells by western blot analysis. Validation of each pathway's involvement in our system will be assessed by genetic knockdown using shRNA for ER stress mediators (PERK& IRE-1) or overexpression of mTOR. Overall, we anticipate that these studies will provide an experimental foundation for the clinical use of vitamin D3 and its analogs in enhancing breast cancer radiotherapy, preventing disease recurrence, and prolonging the life of breast cancer patients.